PRINCIPLES IN MICROBIOLOGY

(SBB 3033)

EXPERIMENT 7:
Antimicrobial analysis

MEMBERS

MATRIC NUMBER

Noor Akmal Binti Azli

D20131063718

Rosurianawatie Binti Mohd Arifin

D20131063722

Siti Nabillah Binti Mohd Zon

D20131963734

Nor Umi Nadia Binti Abdul Rajak

D20131063745

EXPERIMENTS 7 (Antimicrobial analysis)

Objectives
To investigate the degree of antibiotics resistance in a bacterial species using a Kirby-Bauer
test.

ABTRACT
An antimicrobial is an agent that kills microorganisms or inhibits their growth.
Antimicrobial medicines can be grouped according to the microorganisms they act primarily
against. By strict definition, the word antibiotic refers to substances produced by
microorganisms

that

act

against

another

microorganism. In

contrast,

the

term

antimicrobials include all agents that act against all types of microorganisms bacteria
(antibacterial), viruses (antiviral), fungi (antifungal) and protozoa (antiprotozoal). As for this
experiment the antimicrobial agent used is Ampicillin with 100mg/ml followed by Ampicillin
with unknown concentration, bleach, iodine, detergent and alcohol. However, we could said
that all antibiotics are antimicrobials, but not all antimicrobials are antibiotics.

INTRODUCTION

Bacteria have existed long before humans set foot on the earth, and will probably outlive the
human species. They are found on almost every surface available, on our desks, in the food
we eat, and even in and on our bodies. Some bacteria are very helpful, such as E. coli, which
lives in our intestines and helps with digestion and production of Vitamin K. Only a small
number of bacteria are considered harmful and actually cause disease. In this experiment, we
did the antimicrobial analysis. An antimicrobial is an agent that kills microorganism or
inhibits their growth. The main classes of antimicrobial agents are disinfectant, antiseptic and
antibiotic. Disinfectants kill a wide range of microbes on non-living surface or on inanimate
objects to prevent the spread of illness. Many household cleaning agents such as ammonia
and bleach are powerful disinfectants. Antiseptics are applied both on inanimate and animate
object which help in reducing infection. For example in cleaning the cuts and prevent
infection during surgery. Examples of antiseptics include: alcohol, iodine, hydrogen peroxide,
heavy metals such as silver nitrate and detergents. While antibiotic destroy microorganisms
within body. Antibiotics are chemical substances produced by microorganisms that stop the
growth of other microorganisms and are effective in very small doses. Antibiotic production
by microorganisms is common, and most medically valuable antibiotics are produced either
by fungi, especially those in the genus Penicillium, which produces antibiotics such as
penicillin or bacteria in the genus Bacillus, which produce antibiotics such as bacitracin and
polymyxin; or bacteria in the genus Streptomyces, which produce a number of medically
important antibiotics. Antimicrobial agents work in one of two ways: they are either
bacteriostatic or bacteriocidal. A bacteriostatic agent does not kill the bacteria, but inhibits
growth by preventing cell wall formation or interfering with biochemical cellular processes.
A bacteriocidal agent kills bacteria on contact by breaking the protective cell membrane and
lysing the cell, or stopping nucleic acid synthesis, or preventing the bacteria from carrying
out normal chemical reactions. So in the lab we used Ampicillin (unknown concentration),
Ampicilin (100mg/ml), alcohol, iodine, bleach and detergent as an antimicrobial agent. we
will be able to test some common antimicrobials to see how effective they are at stopping
bacterial growth using the zone-of-inhibition method. With this method, a chemical is
applied to a freshly inoculated plate and after incubation, bacterial growth around the
antimicrobial agent is observed. A clear area around the chemical agent (zone-of-inhibition)
indicates that bacteria were killed. The size of the zone is usually proportional to the
effectiveness of the antimicrobial.
MATERIALS

Broth culture of S.epidermis and E.coli

Mueller- Hinton agar
Sterile cotton swab
Forceps
Bunsen burner
Antibiotics dics (Ampicilin, tetracyclin, Erthromycin, Penicilin)
Alcohols, iodine, bleach,detergent
Whatman filter paper

METHODOLOGY:
1. The contents of broth culture of S. aureus is swirled until it is equally
murky throughout.
2. Cotton swab was used to created a lawn of S. aureus on 2 plates of
Mueller-Hinton agar aseptically.
3. Antibiotics disc was picked by using sterile forcep and was placed on
opposites sides of petri dish containing S. aureus. Antibiotics disc
was tap gently to stick them to the agar.
4. Step 1-3 was repeated using plates of E. coli.
5. All the plates was wrapped by using Parafilm and the plate was
placed in incubator at 37C for 24 hours.
6. We checked the presence of antibiotic activity after 24 hours in
incubator. We observed for a clear area, called zone of inhibition
surrounding a paper disc.
7. We measured the diameter (mm) od the zone of inhibition to
determined the affectivity of an antibiotic.
8. The test was repeated by using alcohol, iodine, bleach and
detergent.

RESULTS ( Noor Akmal & Siti Nabillah)

Ampicillin
100mg/ml

Ampicillin
unknown

Negative control

Alcohol

Figure 1 : Cultures of S. aureus in presence of antimicrobial agent that is

ampicillin (unknown concentration), ampicillin 100mg/ml, alcohol and
negative control.

Bleach

Detergent
Iodine

Negative control

Figure 2 : Cultures of S. aureus in presence of antimicrobial agent that is

iodine, bleach, detergent and negative control.

Alcohol
Ampicilin
unknown
Negative
control

Ampicilin
100mg/ml

Figure 3 : Cultures of E. coli in presence of antimicrobial agent that is

ampicillin (unknown concentration), ampicillin 100mg/ml, alcohol and
negative control.

Bleach

Detergent

Iodine

Negative
control
Figure 4 : Cultures of E. coli in presence of antimicrobial agent that is
iodine, bleach, detergent and negative control.

Results for S. aureus sp

Antimicrobial Agent

Diameter

zone

inhibition

of (Condition of bacteria)
Sensitive (S)
Intermediate (I)

Negative control
Ampicillin

of

Resistance (R)
-

unknown 2.0

Sensitive

concentration
Ampicillin 100mg/ml

1.8

Intermediate

Alcohol

1.7

Intermediate

Iodine

1.5

Intermediate

Bleach

1.6

Intermediate

Detergent

2.0

Sensitive

Table 1: Antimicrobial analysis on Staphylococcus aureus

Results for E.coli sp

Antimicrobial Agent

Diameter
inhibition

zone

of (Condition of bacteria)
Sensitive (S)
Intermediate (I)
Resistance (R)

Negative control
Ampicillin

of

unknown 3.2

Sensitive

concentration
Ampicillin 100mg/ml

3.4

Sensitive

Alcohol

Resistance

Iodine

1.4

Intermediate

Bleach

2.3

Sensitive

Detergent

Resistance

Table 2: Antimicrobial analysis on E.coli sp

DISCUSSION: (Noor Akmal & Siti Nabillah)

In this experiment, we want to determine the degree of antibiotic
resistance in S. aureus and E. coli by using Kirby-Bauer test also called a
zone of inhibition test. This method is used to measure antibiotic
resistance and to test the ability of antibiotic to inhibit microbial growth.
In this experiment, the broth culture of S. aureus and E. coli are spread
over an agar plate using a sterile swab, then the antibiotic disc incubated
in the presence of antimicrobial agent that is ampicillin unknown
concentration, ampicillin 100mg/ml, alcohol, iodine, bleach and detergent
and also negative control. Negative control can differentiate between test
failure or invalid test by observing negative control. No growth should be
there in negative control. Upon incubation, antibiotic diffuses from the
disk into the surrounding agar. If sensitive to the antibiotic, the bacteria
will be unable to grow in the area surrounding the disk called a zone of
inhibition.

The size of this zone is dependent on a number of factors,

including the sensitivity of the bacteria

to the antibiotic, the rate of

diffusion of the antibiotic through the agar, and the depth of the agar.
Bacteria that are resistant to an antibiotic will not show a zone of
inhibition.
Based on the results of this experiment, for S. aureus sp, it shows
that the bacteria is sensitive in antimicrobial agent of ampicillin of
unknown concentration and detergent because diameter zone of inhibition
is longer compared to other antimicrobial agent that is iodine, bleach,
alcohol and ampicillin 100mg/ml. The diameter of zone of inhibition of
ampicillin of unknown concentration and detergent is 2.0mm. From the
result, we could see that zone of inhibition for

ampicillin of unknown

concentration and detergent is more higher compared to others. S. aureus

sp is intermediate in iodine, bleach, alcohol and ampicillin 100mg/ml . The
diameter of zone of inhibition of iodine, bleach and alcohol

is 1.5mm,

1.6mm, 1.7mm and 1.8mm.

For E.coli sp, the results

shows that the bacteria is resistance in

antimicrobial agent of alcohol and detergent because it do not show a

zone of inhibition compared to other antimicrobial agent that is iodine,

bleach, ampicillin 100mg/ml and ampicillin of unknown concentration that
have diameter zone of inhibition. The bacteria is sensitive in antimicrobial
agent of ampicillin of unknown concentration, ampicillin 100mg/ml and
bleach. The other antimicrobial agent that is intermediate was iodine.
Determination of

bacteria whether it is sensitive, resistance or

intermediate was based on the diameter zone of inhibition from the

results that we obtained.
detergent is 0mm.

Diameter zone of inhibition for alcohol and

The bacteria was able to grow on the agar plate in

this condition. Diameter zone of inhibition for ampicillin of unknown

concentration, ampicillin 100mg/ml and bleach is 3.2mm, 3.4mm, 2.3mm.
The surrounding area that the bacteria unable to growth is higher in the
plate. It shows that the bacteria is sensitive towards this antimicrobial
agents. Iodine shows it is

intermediate based on

diameter zone of

inhibition that is 1.4mm.

Based on the results, E.coli sp is resistant and sensitive to a larger
number of antimicrobial agent compared to S. aureus sp. From this
experiment, we can also able to know the factors that affect diameter
zone of inhibition. Concentration of the antibiotic on the disks is an
important factor as well as the size of the affected bacterial colonies.
Other than that, the temperature of incubation, the length of incubation,
the thickness of the agar in the plates, the age of the culture used to
create the lawn, the density of the culture used to create the lawn, the
age of the antibiotic disks, and the laboratory technique of the student
when conducting the experiment also influence in the results.

RESULT (Nor Umi Nadia & Rosurianawatie)

C:
Detergent

A: Negative
control

D: Iodine
B: Bleach

Figure 5: Zone of inhibition of Escherichia coli treated with (A) Negative control, (B)
Bleach, (C) Detergent and (D) Iodine.

E: Ampicillin
100mg/ml
G: Negative
control
F: Alcohol

H: Ampicillin
(unknown)

Figure 6: Zone of inhibition of Escherichia coli treated with (E) Ampicillin 100mg/ml, (F)
Alcohol, (G) negative control and (H) Ampicillin (unknown concentration)

Antimicrobial agent

Diameter zone of

Condition of bacteria

inhibition
Negative control
0
Ampicillin (unknown concentration)
3.0
Sensitive
Ampicillin 100mg/ml
3.1
Sensitive
Alcohol
0
Resistance
Iodine
1.0
Intermediate
Bleach
2.4
Sensitive
Detergent
0
Resistance
Table 3: Antimicrobial analysis on Escherichia coli

C: Negative
control

A: Bleach

D: Detergent
B: Iodine

Figure 7: Zone of

inhibition of

Staphylococcus aureus treated with (A) Bleach, (B) Iodine, (C) Negative control and (D)
Detergent.

G: Negative
control

E: Ampicillin
100mg/ml

H: Ampicillin
(unknown)

F: alcohol

Figure 8: Zone of inhibition of Staphylococcus aureus treated with (E) Ampicillin 100mg/ml,
(F) Alcohol, (G) negative control and (H) Ampicillin (unknown concentration)

Antimicrobial agent

Diameter zone of

Condition of bacteria

inhibition
Negative control
0
Ampicillin (unknown concentration)
2.5
Sensitive
Ampicillin 100mg/ml
2.4
Sensitive
Alcohol
0
Resistance
Iodine
1.5
Intermediate
Bleach
1.8
Intermediate
Detergent
2.1
Sensitive
Table 4: Antimicrobial analysis on Staphylococcus aureus

DISCUSSION (Nor Umi Nadia & Rosurianawatie)

In this experiment, we did an antimicrobial analysis on 2 types of bacteria which are
Escherichia coli and Staphylococcus aureus. The antimicrobial agents that we used are used
Ampicillin (unknown concentration), Ampicilin (100mg/ml), alcohol, iodine, bleach and
detergent. We used 2 agar plate and each plate were divided into 4 sections (refer to results).
Before we put the antibiotic discs that were treated with the antimicrobial agents following
the section, we used the sterile cotton swab to create a lawn of Escherichia coli and
Staphylococcus aureus on the 4 different agar plates. This is to make sure all the agar surface
are covered with the bacteria. Then the antibiotic disc that treated with antimicrobial agents is
placed in each section. After that the agar plate left incubated for 24 hours.
After 24 hours, the result came out. We measure the diameter of zone of inhibition for
every section and tabulated it. Zone of inhibition is a clear area around the chemical agent
which indicates that bacteria were killed. For the antimicrobial analysis for Escherichia coli,
the most sensitive agent is Ampicillin with 100mg/ml which the diameter of the zone of
inhibition is 3.1 then followed by Ampicillin with unknown concentration which the diameter
of zone of inhibition is 3.0. Both of these antimicrobial agents are antibiotic. For the bleach,
the diameter of zone of inhibition is 2.4 which indicate that the bacteria are sensitive towards
the bleach.

For alcohol and detergent, the diameter of zone of inhibition for both are 0. The
Escherichia coli were resistance toward both antimicrobial agents. The significance of this
may be because of survival of E.coli in the presence of ethanol. For the detergent the bacteria
should be inhibited because the detergent is an antiseptic. For the iodine the diameter of zone
of inhibition are 1.0 which indicate the bacteria is intermediate towards it. As we know the
diameter of size of the zone is usually proportional to the effectiveness of the antimicrobial.
So from the result for antimicrobial analysis for Escherichia coli we can see the strongest
antimicrobial agent is Ampicillin with 100mg/ml followed by Ampicillin with unknown
concentration, bleach, iodine, detergent and alcohol.
As for Staphylococcus aureus, the most sensitive agents is Ampicillin with unknown
concentration which the diameter of zone of inhibition is 2.5 the followed by the Ampicillin
with 100mg/ml which the diameter of inhibit zone is 2.4 which is contradict from the
Escherichia coli.For the bleach, the diameter of zone of inhibition is 2.1 which indicate that
the bacteria are sensitive towards the bleach.
Then, for alcohol, the diameter of zone of inhibition is 0. The Staphylococcus aureus
were resistance the antimicrobial agents. We could said that the Staphylococcus aureus can
survive in the presence of ethanol same as the Escherichia coli. For the detergent which the
diameter of zone of inhibition is 1.8 followed iodine the diameter of zone of inhibition are 1.5
by which indicate the bacteria is intermediate towards it. Thus, from the result, we could see
that antimicrobial analysis for Staphylococcus aureus that the strongest antimicrobial agent is
Ampicillin with unknown concentration followed by Ampicillin with 100mg/ml, detergent ,
iodine ,bleach, and alcohol.

CONCLUSION
From this experiment we could conclude that there is several
antimicrobial agent for the microbes to be able resist them from being
killed. In this experiment we can see that the most effective microbial
agents is ampicillin 110mg/ml while the most least effective agent is
alcohol for the E.coli.

Meanwhile for the S.aures

we can see that the

most effective is detergent and the least effective is alcohol. All the
conclusion is based from the observation the result shown. We use the

Kirby Bauer test which is a antibiotic testing (KB testing or disc diffusion
antibiotic sensitivity testing) which uses antibiotic-impregnated wafers to
test whether bacteria are affected by antibiotics. However we can that all
antibiotics are antimicrobial but not all antimicrobial are antibiotics.

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Microbiology

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC88911/
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